1. Representations of Odor in the Piriform Cortex
Dan D. Stettler, Richard Axel 
Neuron 
Volume 63, Issue 6, Pages (September 2009)
DOI: /j.neuron
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
Odorant-Evoked Responses in Mouse Piriform Using In Vivo Two-Photon Calcium Imaging
(A) Views of the lateral (top) and ventral lateral (bottom) surface of the mouse cerebral hemisphere showing the piriform cortex outlined by a white dotted line. The imaged area is highlighted in blue. The same region of piriform was imaged in different animals using the middle cerebral artery (MCA) and lateral olfactory tract (LOT) as landmarks. Scale = 1 mm.
(B) Coronal slice of piriform showing the characteristically dense layer 2, which is occupied primarily by the somata of pyramidal cells that receive direct input from the bulb upon their apical dendrites in layer 1. Neuronal nuclei were labeled using NeuroTrace. Scale = 100 μm.
(C) Baseline fluorescence of cells in layer 2 loaded with Oregon Green 488 BAPTA-1 AM and imaged in vivo with two-photon microscopy. Scale = 50 μm.
(D) The cells in panel (C) were identified by custom software using their baseline fluorescence and were overlayed with cellular responses to odorants presented in gradations of red corresponding to the level of response. Di(propylene glycol) (DPG) is used to dilute odorants to approximately equal concentration (3 ppm in air). Five structurally diverse odorants, including the complex odorant, female mouse urine, activate dispersed subsets of cells. 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a component of fox urine that elicits innate aversive behavior in the mouse, also activates cells in this region.
(E) Traces depicting changes in fluorescence elicited by the five odorants for the five cells outlined in (C). Adjacent cells can respond selectively to different odorants. The traces of odorants not eliciting a response are gray, and those of DPG and air are blue.
Neuron  , DOI: ( /j.neuron )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. Figure 2
Odorant-Evoked Calcium Responses in Piriform Cortex Are Highly Consistent
(A) Responses to DPG and hexanal are superimposed upon an image of identified cells. Fifty-five of 335 cells in this field exhibit significant increases in fluorescence when the animal is stimulated with hexanal. Scale = 50 μm.
(B) The superimposed traces from 20 sequentially delivered odorant pulses measured using a photo-ionization detector are shown at the top in blue. The superimposed fluorescence traces from 20 hexanal (red) or DPG (blue) trials are presented below for six different cells along with the cross-trial average for each cell. The individual trial traces are highly consistent, even for those cells with small responses.
(C) The average ΔF/F for the 2 s epoch after the onset of hexanal (red) or DPG (blue) delivery is shown for all 20 trials for the 55 cells identified as responding to hexanal. Single-trial hexanal responses are distributed monomodally around the cross-trial average, shown in pink.
Neuron  , DOI: ( /j.neuron )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3 Concentration Dependence of Odor-Evoked Responses
(A) Graded maps showing responses to octanal at concentrations (in ppm) indicated in the upper right corner of each panel. At 3.3 ppm, 12% of the cells are activated, and this value only increases to 20% at 82.5 ppm, a 25-fold higher odorant concentration. Scale = 50 μm.
(B) Most cells exhibit nonlinear increases in intensity with increasing odorant concentration, but one-third of the cells exhibit a decline in activity at higher concentration.
(C) The frequency of responsive cells is nonlinear as a function of octanal concentration and saturates at a value of 20% active cells.
Neuron  , DOI: ( /j.neuron )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4
Odorants Evoke Responses in Unique but Overlapping Ensembles of Piriform Neurons
(A) Cells responsive to octanal (red) overlayed on the responses to five other odorants (green) in piriform layer 2. The cells are presented as either responsive or not responsive with no graded color code. Yellow cells respond to both odorants. Each odorant evokes responses in different but overlapping ensembles of piriform cells. Scale = 50 μm.
(B) Array showing the responses of cells from panel (A) to each of the six presented odorants. Only those cells responding to at least one odorant are shown. A yellow dot in the upper left corner of the cell/odorant pairing indicates a significant response. Approximately one-quarter of the cells respond to more than one odorant, but only six of the 85 cells respond to three or more odorants.
(C) Octanal (red) and α-pinene or hexanal (green) responsive cells in multiple adjacent imaging sites containing 972 labeled cells in layer 2 of the piriform cortex. The imaged sites are superimposed upon the blood vessels at the surface of the piriform that are used for alignment. Of the 88 cells responding to octanal, nine also respond to the structurally dissimilar odorant α-pinene, and 19 also respond to the highly similar odorant hexanal. Scale = 100 μm.
(D) Sample fluorescence traces for four cells from panel (C). Some cells respond strongly to octanal (top) or hexanal without responding even weakly to the other.
Neuron  , DOI: ( /j.neuron )
Copyright © 2009 Elsevier Inc. Terms and Conditions

6. Figure 5
Distributed Odorant Representations Extend across Wide Regions of Piriform Cortex
(A) Ventral lateral view of a mouse cerebral hemisphere superimposed with the imaging craniotomy. The surface vasculature enables the montage of imaging sites positioned widely across the piriform and the determination of their location within the hemisphere. Scale = 1 mm.
(B) Montage of images showing the baseline fluorescence of labeled cells in five contiguous imaging sites across layer 2 of the piriform cortex in the hemisphere shown in panel (A). The montage overlies the vessels at the surface of the cortex that was used for alignment. The imaged region contains 1301 loaded cells. Scale = 200 μm.
(C) Responsive cells to four odorants across a wide region of piriform cortex shown in panel (B). The odorants were diluted to approximately the same concentration (3 ppm in air) except for α-pinene, which was presented at 15 ppm. Different odorants elicit different numbers of responsive cells. The density of responsive cells to a given odorant can vary three-fold over ranges of 100 μm.
Neuron  , DOI: ( /j.neuron )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. Figure 6
The Response of Piriform Cells to a Mix of Odorants Exhibits Strong Suppression and Weak Synergy
(A) Montage of four imaging sites from layer 2 of piriform cortex containing 1039 labeled cells. The red cells in the upper left panel respond to either octanal or α-pinene presented alone. The green cells in the upper right panel respond to a mix of octanal and α-pinene at the same concentration as used at the left. The overlap between the responses to the individual odorants and to the mix is shown in the lower left. Cells responsive to individual odorant alone and the mix are yellow. Cells responsive to odorant presented alone but not to the mix remain red, whereas cells responsive to the mix but not individual odorants are green. 36% of the 188 cells responsive to the individual odorants do not respond to the mix. The difference between the sum of the responses to the individual odorants and the response to the mix, with gradations of red indicating higher mix responses and gradations of blue indicating lower mix responses, is presented in the lower right. Scale = 100 μm.
(B) Sample traces from panel (A) showing a cell that exhibits a similar response to α-pinene and the mix (top), cells that exhibit a partially (second from top) or completely (third from top) suppressed response to the mix, and a cell that responds only to the mix (bottom). Cellular responses to mix alone are invariably weak.
(C) Ranked-ordered responses to octanal (red) and responses to the mix of octanal and α-pinene (green) shown for those cells that responded to octanal but not α-pinene. Data presented are for 2177 cells from the imaged fields shown in panel (A), as well as three additional, nonadjacent fields in the same animal.
(D) The sum of each cell's response to the individual odorants presented separately compared with the cell's response to the mix.
Neuron  , DOI: ( /j.neuron )
Copyright © 2009 Elsevier Inc. Terms and Conditions